Study On Preparation Of Gel/Odex/Apo Hydrogel And Its Skin Wound Healing Effect In Mice

Acute skin injury is one of the most common clinical diseases.Diabetes,kidney infection,foreign bodies,malnutrition,immune deficiency,aging and other factors will affect the normal healing process of wounds,thus impeding the recovery of tissues,causing wound inflammation and skin ulceration,leading to long-term medical burden and decreasing quality of life for patients.Hemostatic sponges,gauze,hydrogels and bandages are widely used as traditional effective hemostatic products in clinical applications.However,dry wound dressings tend to adhere to the damaged surface of the skin wound,which cause secondary trauma after removal.Hydrogels have unique characteristics such as high water content,sufficient flexibility,elasticity,biocompatibility and high sensitivity to physiological environment,which are considered to be the best candidates for wound dressings.Recently,a considerable amount of research has been devoted to the development of hydrogels to promote wound healing.Among them,injectable hydrogels have attracted extensive attention.Gelatin,as a collagen derivative,has non-immunogenic properties,cell adhesion behavior and coagulation properties,which can be used as the main raw material for wound dressing.Dextran is a kind of organic compounds with biocompatibility,non-toxicity and no immunogenicity.Oxidized dextran can be used in the modification of gelatin hydrogels to enhance its stability.Apocynin is a NADPH oxidase inhibitor that blocks the production of reactive oxygen species（ROS）and promotes wound healing.Hydrogels prepared by oxidized dextran,gelatin and apocynin can provide three-dimensional support for cell growth,and provide a good transport channel for cell gas exchange,nutrient transport and metabolic products excretion.Therefore,this paper prepared Gel/Odex/Apo injectable hydrogel and studied its therapeutic effect on skin injury in mice,which is expected to provide an experimental basis for the clinical application of hydrogel in the treatment of acute skin injury.ObjectivesIn this study,an injectable hydrogel（Gel/Odex）was prepared by the reaction of gelatin and oxidized dextran through Schiff base,and anti-inflammatory drug Apo was loaded to further obtain multifunctional hydrogel（Gel/Odex/Apo）.The effects of Gel/Odex/Apo hydrogel on wound repair in mice were studied by physical and chemical properties characterization in vitro and biological evaluation in vivo and in vitro.MethodsPart I:Preparation,properties and characterization of Gel/Odex hydrogels¹H NMR,FTIR and hydroxylamine hydrochloride titration were used to qualitatively and quantitatively determine the success of Odex.Three groups of hydrogels,named Gel/Odex_0.3,Gel/Odex_0.5,Gel/Odex_0.7,were prepared by Schiff base reaction,and the gelation time was determined by tube inversion method.The water content of hydrogel was detected by freeze-drying method.Scanning electron microscope（SEM）was used to observe the surface and internal structure of hydrogels.Mechanical properties of hydrogels were measured by rotating rheometer.The degradability of hydrogels in vitro was detected by weighing method.CCK-8 and AM-PI double fluorescence staining were used to detect the effects of three kinds of hydrogel extracts on the survival of HaCaT and L929 cells at 1 and 3 days.The hemolysis and coagulation of hydrogel were detected by hemolysis test and dynamic coagulation test respectively.A group of hydrogels was optimized for subcutaneous injection of mice,and the histocompatibility of the hydrogel was detected by hematoxylin-eosin staining on days 3,7 and 14,and the hemostatic property of the hydrogel was detected by hepatic hemorrhage model of mice.Part II:Effects of Gel/Odex/Apo hydrogel on skin injury repair in miceCCK-8 was used to detect the effects of different concentrations of Apo drug on the toxicity of HaCaT and L929 cells.The cell compatibility of HaCaT and L929 in Gel/Odex and Gel/Odex/Apo hydrogels after Three-dimensional culture of 1 and 3 days was detected by double fluorescence staining with AM-PI.The drug sustained release of Gel/Odex/Apo hydrogel was detected by Apo drug release.Kunming mice（male）aged from 5 to 6 weeks were used as experimental animals.The injury model of mice was established by full-thickness skin resection method,which were randomly divided.The wound healing and the area of wound were observed at 0,3,7 and 14 days after treatment.On the 14th day of healing in the treatment group,the wound skin tissue of mice was removed.Granulation tissue and epidermis formation were detected by HE staining.Masson staining was used to detect new collagen deposition.The expression ofα-SMA and MMP-9 were detected by immunohistochemical staining.The expression of CD31 and PCNA in wound tissue was detected by immunofluorescence staining.The expression of IL-6 and TNF-αrelated proteins was detected by Western blot.ResultsPart I:Preparation,properties and characterization of Gel/Odex hydrogelsThe results of ¹H NMR and FTIR showed that the characteristic peaks of aldehyde group appeared in Odex compared with Dex,which proved that the oxidation of dextran was successfully prepared.The oxidation rate of Odex was 70.5%.Gel/Odex hydrogel was injectable and can be shaped according to the mold,which can better fill the damaged parts of the skin.SEM results showed that the surface and interior of hydrogel are three-dimensional porous network structure.The gelation time of Gel/Odex hydrogels was 6-12 min,and the water content was higher than 95%.With the increase of Odex concentration,the gelation time and in vitro degradation rate decreased and elastic modulus of hydrogels increased.Gel/Odex hydrogel had good cell compatibility,blood compatibility and promoting coagulation.There is no statistical difference between the three groups of hydrogels.According to the degradation performance and gelation time of hydrogels,Gel/Odex_0.5 hydrogel was used for subsequent experiments.The mice were injected subcutaneously with Gel/Odex_0.5 hydrogel,and no obvious inflammatory reaction was found by HE staining at 3,7 and 14 days,indicating good histocompatibility.The hemostasis effect of Gel/Odex_0.5 hydrogel was proved by hepatic hemorrhage model in mice.Part II:Effects of Gel/Odex/Apo hydrogel on skin injury repair in miceCCK-8 results showed that 20 mg/L Apo had no toxic effect on HaCaT and L929cells.The three-dimensional culture of HaCaT and L929 in Gel/Odex/Apo hydrogel showed good cytocompatibility,which could continuously release Apo.The model of full-thickness skin excision in mice was established successfully.After 14 days of treatment,compared with Normal saline,Tegaderm?,Gel/Odex and Gel/Odex/Apo groups all promoted wound healing,but Gel/Odex/Apo significantly reduced the wound area.HE staining showed that Gel/Odex/Apo had a more obvious effect on wound healing,and the formation of upper epidermis and granulation tissue were more complete.Masson staining showed that collagen deposition in Gel/Odex/Apo group was significantly increased.Immunohistochemical results showed that the expression of inflammation-related protein MMP-9 was decreased and the production of vascular smooth muscle actinα-SMA was increased in Gel/Odex/Apo group（P<0.05）.Western Blot results showed that the expression levels of inflammation-related proteins IL-6and TNF-αwere significantly decreased in Gel/Odex/Apo group.Immunofluorescence results showed that the expression of endothelial marker CD31 and PCNA increased in Gel/Odex/Apo group（P<0.05）.Conclusion1.Gel/Odex hydrogel had injectability,biodegradability,self-healing,high moisture content,good mechanical properties,etc.The surface and interior of the hydrogel had uniform pore shape and three-dimensional network structure,and it had good blood compatibility and can promote blood coagulation.Gel/Odex_0.5 had good histocompatibility and hemostasis properties,and the gelation time can meet the operability of animal experiments.2.Gel/Odex/Apo can significantly improve the ability of wound repair in mice with skin injury and promote the formation of tissue in skin injury area.The potential mechanism may be that Gel/Odex/Apo can achieve the effect of wound healing by reducing wound inflammatory response,promoting cell proliferation,enhancing upper epidermal regeneration and collagen deposition,and promoting angiogenesis.